预测环境因子潜在致癌性方法的探讨
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摘要
癌症已是人类健康的最大威胁之一,尽管其发生、形成机理仍未被完全阐明,但大部分癌症由环境因子引发已属共识。某些环境因子引起细胞生长因子及其受体表达水平的改变,从而激发、增加DNA突变率并导致癌细胞的发生。因此,通过检测环境因子对DNA突变率的激发结果来估计其诱发癌症的可能性已成为一项重要的研究内容。从数学分析方法上来看,它属于统计推断范畴,即由一系列实验结果、数据来估计、推断随机事件发生的可能性;而正确的检测、分析方法则是降低推断分析中“以真作假”和“以假作真”等错误机率的关键。
Ames测试是通过检测化合物能否增加组氨酸缺陷型沙门氏菌的回复突变来推测其潜在致癌性,该测试对化合物的致突变性、潜在致癌性易于测试、判断;但对于含组氨酸的复杂混合物,经典的Ames测试并不适合,检测、分析方法的不当即会造成“以假作真”的错误推断。近年来,在检测中药制剂的致突变性时,这一问题又恰恰发生了;当然,这类问题同样存在于用Ames测试来检测其它复杂混合物的致突变性时。
不久前,国家发展改革委发布了《高技术产业化“十一五”规划》,规划提出在“十一五”期间我国将实施16个高技术产业化重大专项,其中包括“现代中药”重大专项。然而,能否准确地检测、评定中药的致突变性、潜在致癌性会直接影响国家对中药产业的政策,同时也关系到中药能否被充分利用,更是关系到人类健康的头等大事。因此,建立一个适于分析复杂混合物致突变性的方法就显得尤为迫切和重要。
以表型为检测指标的突变检测方法往往会受到多种因素的影响,同时环境因子对DNA的致突变作用也绝不仅仅局限于某个特定的基因(如Ames测试中组氨酸操纵子的某个基因),这涉及到随机性还是特异性的问题,这一点在理论上至今还没有确切的答案;而直接以DNA为检测指标的突变检测方法应是解决这类问题的关键所在。本论文的相关研究工作即在上述背景下展开,三年来取得了以下研究成果。
一、对影响Ames测试结果不确定性因素的分析
Ames测试是目前各个实验室普遍采用的检测环境来源的致突变物、潜在致癌物的通用初筛方法之一,但该测试在实验设计、实验过程及实验结果的分析上仍存在一些不确定性的因素和疑问。评估一个微生物群体的自发突变率和诱发突变率在遗传理论和实验技术上都是一个涉及多方面因素的复杂性难题,估计诱发突变率时涉及到对两个正态分布的混合分布参数的估计,它需要大量的样本,但Ames测试中样本数均较小,无法用条件矩等方法估计混合分布的参数;DNA发生突变的随机性和诱发因素的相关性也是尚未阐明的难题之一。
1.1总体上来说,Ames测试菌株只能检测出导致小规模突变的致突变物和理化因子,对于更多其它类型的致突变物和理化因子则无法检出,这就导致了该测试在有效检出的致突变物类型及种类上存在较大的局限性,且易出现假阴性结果。
1.2 Ames测试菌株只能检测出发生在特定位点上的突变,发生在其它位点上的突变则无法检出。
1.3 Ames测试建立在营养贫瘠的培养基上,当用其检测复杂混合物如中药的致突变性时,可能产生假阳性结果,适应性突变更加剧了这种可能性。
1.4 Ames测试中可统计的回复子菌落数是培养时间的函数,肉眼可见的回复子菌落数随培养时间的延长而增多,人为规定的48 h或其它时间是一个“约定俗成”的惯例,缺乏其应具有的科学内涵。
1.5 Ames测试中各类型的回复子在选择培养基上的生长速率各不相同。在统计时,相当数量的生长慢的回复子因未能形成肉眼可见的回复子菌落而不能被统计,这也是Ames测试这类基于试验组与对照组间回复子菌落数差异的大小来判定物质致突变性的检测方法的致命缺陷之一。
分子生物学技术的发展使直接以DNA为检测指标的物质致突变性检测方法得以实现,其可避免Ames测试中较多不确定性因素和结果。
二、建立了一个检测中药制剂潜在致癌性的方法
Ames测试建立在含微量组氨酸的固体培养基上,要求试验组与对照组中组氨酸含量相同,以使得试验组与对照组中测试菌的分裂次数、生长量均趋于相同。该测试在检测中药致突变性时,因受中药中组氨酸含量的影响,试验组中组氨酸含量高于对照组而使对照组失去了可比性,致使多种中药在该测试中呈阳性。本研究用Ames测试检测了7种中药的致突变性,所有测试的中药均呈阳性。
Ames测试菌株在LB培养基中培养时,其生长量及瞬时生长速率不受培养基中组氨酸浓度的影响;处于衰亡期的Ames测试菌株TA100和TA98的相对回复突变频率与测试体系中诱变剂的浓度间存在剂量效应关系。基于上述的结果,建立一个根据试验组与对照组中衰亡期的测试菌的相对回复突变频率的差异性来评定物质致突变性的方法。中药所含的组氨酸不会影响该测试结果的准确性,因而适于检测中药的致突变性,进而推断中药的潜在致癌性。
通过测定中药中组氨酸含量并结合统计学分析,证实这些中药的“致突变性”源于中药所含组氨酸导致的假阳性结果。应用新建方法测定了在Ames测试中呈“阳性”中药的致突变性,在该测试中试验组中测试菌的相对回复突变频率与中药浓度间不存在剂量效应关系,并且试验组与阴性对照组中测试菌的相对回复突变频率无显著性差异(t-test;p＜0.05),说明这些中药在该测试中呈阴性,即没有致突变性,进而推断这些中药没有潜在致癌性。
三种中药(银杏叶、猫眼草、三棱)进行了哺乳动物骨髓细胞染色体畸变试验,结果显示所测试中药均无致突变性,该测试结果进一步佐证了新建方法检测中药致突变性的有效性,也说明该方法适于评价中药的潜在致癌性。
三、改进了应用MutS蛋白检测突变的方法
MutS蛋白在体外能识别并结合含有错配碱基的DNA片段,多种基于此属性的突变检测方法应运而生。本文中实验结果证实,MutS蛋白在高剂量下亦能很好地结合纯合双链DNA,并且这种结合具有剂量依赖性;在低剂量下,MutS蛋白结合杂合双链DNA的能力显著强于其对纯合双链DNA的结合。
本研究中摒弃了测序法和代表性差异分析法,而采用能有效区别样品间DNA量的微小差异的实时荧光定量PCR技术来检测突变,直接比较实验样品与对照样品的Ct值的差异来判断、检测突变。实时荧光定量PCR技术的应用大大简化了检测突变的过程。
另外,证实了用此种方法能够检测在背景细胞中低至2%的突变体,是一种改进的、简便的、在DNA水平上检测突变的方法。
四、极低比例的未知突变体检测方法的探讨及初步建立
多种检测DNA片段多态性和突变的方法例如测序法及单链构象多态性(SSCP)只能检测出群体中高比例的突变体(＞9%),对于更低比例的突变体则无法有效检出。
用于检测低比例的突变体的方法需要具有高分辨力和高灵敏度,目前满足这两个条件的检测方法如芯片、生物电极等又因成本过高而难以被普遍地应用。本文设计了一个可提高群体中极低比例的突变体的相对及绝对含量的PCR程序,应用此程序提高了突变体的相对及绝对含量后,用激光诱导荧光毛细管电泳-单链构象多态性分析了实验样品及对照样品经PCR扩增后的产物。实验结果显示,在毛细管电泳谱图中,实验样品的扩增产物比对照样品明显地多出了一个峰,此峰是实验样品中极低比例的未知突变体(10~(-5))经扩增后被检测到。此方法具有一定的可行性,但还需进一步优化条件以提高突变体的检出效率,在此基础上建立能直接根据DNA的变化来检测物质致突变性的方法。
Cancer has been one of the greatest threats to human health;despite the mechanism of its occurrence and formation has not yet been fully explained,a consensus has been already obtained that most cancers are caused due to some environmental factors. Some environmental factors can change the expression levels of some cell growth factors and their receptors,which leads to the increase in mutation rates of DNA and occurrence of cancer cells.Therefore,the investigation on the ability of environmental factors to induce gene mutation is an effective way to estimate their potential to cause cancer.From the mathematical point of view,this involves the probability and statistical inference,that is,the possibility of an incident is estimated based on a seriety of experiment results and data.It is crucial for decreasing the chance of misjudgment to adopt the appropriate detection and analysis methods,and the results from inappropriate methods will inevitably lead to wrong misjudgments.
The Ames test is based on the principle that mutagenic compounds can increase the reversion frequencies of histidine-deficient strains,thereby speculating their potential carcinogenicity.The potential carcinogenicity of pure compounds can be well estimated with the Ames test;however,as to complex mixtures,it is inappropriate to adopt the Ames test to monitor their potential carcinogenicity.Similarly,it is inappropriate to adopt the Ames test to monitor the potential carcinogenicity of traditional Chinese medicine because traditional Chinese medicine is a mixture,too.
Not long ago,the National Development and Reform Commission issued the high-tech industries development during 11th five-year plan,16 major projects including the 'modem traditional Chinese medicine' project will be implemented.So, whether the mutagenicity of traditional Chinese medicine can be accurately determined will have direct affects on not only the national policy on the traditional Chinese medicine industry,but also the utilization of traditional Chinese medicine. Therefore,it is necessary to develop a novel mutagenicity detection method for the complex mixtures including traditional Chinese medicine.
The bacterial reversion assays such as the Ames test were affected by multiple factors;at the same time,the environmental factors can change not only a specific gene but also many other genes,which is related to specificity or random.Therefore, the mutation detection method at DNA level may be a way to solve the above issues.
The study in this paper started with these problems mentioned above,and the following results were obtained in the past three years.
1.Analysis of the factors leading to uncertainty of Ames test results
The Ames test is used world-wide as an initial screen to determine the mutagenic potential of new chemicals and drugs;however it involves many uncertain factors. The estimation of the spontaneous and induced mutation rate of a microbial group is complex from the point of view of the genetic theory and experimental technique, which involves a number of factors.The estimation of induced mutation rate is related to the mixing of two normal distributions,which needs lots of samples;unfortunately, the number of samples in the Ames test is small,as a result the parameter of the mixed distribution can not be estimated with conditional moment closure model.
1.1 Only small-scale mutations such as base substitution and one or two nucleotides
insertion or deletion can be detected by the tester strains used in the Ames test,as a result many other types of mutation can not be monitored.
1.2 Only the mutations occurring at specific locuses such as hiG46 locus can be monitored,many more mutations occurring at other locuses can not be detected with the Ames test.
1.3 The Ames test is based on the minimal medium,which made this method unsuitable for the detection of mutagenicity of nutrient-rich materials. Additionally,this barren environment might also lead to the adaptive mutation, which much more enhanced the uncertainty of test results.
1.4 The revertant colony numbers are the function of incubation time,so the time to count the revertant colony numbers(48 h) is relatively arbitrary,lacking of scientific connotation.
1.5 Various types of revertants grew at different rates in the selective medium; thereby the revertants with a higher growth rate were probably counted compared with those with lower growth rates.That is to say,not all the revertants could be counted,which was a vital defect to the Ames test.
2.A novel potential eareinogenieity detection method for traditional Chinese medicine
The Ames test is based on the test system that contains trace amounts of histidine (15.52μg/plate),and the amounts ofhistidine in the test groups and the control groups are same.Many traditional Chinese medicines were estimated to be mutagenic with the Ames test,but these results on the mutagenicity of these traditional Chinese medicines might be questionable due to considerable amounts of histidine in them. Several traditional Chinese medicines were estimated with the Ames test,and all of them were found to be mutagenic.However,these samples were nonmutagenic after deducting the revertant numbers resulting from the histidine in the samples.
Additional excessive external histidine did not affect the growth of TA100 when TA100 was cultured in LB broth;furthermore,dose-response relationships occurred between the mutagen concentrations and the relative reversion frequencies of the tester strains during the declining phase.According to these results,a novel strategy based on liquid LB broth to estimate the mutagenicity of samples containing much free and/or protein-bound histidine was developed.This strategy could be expected to avoid the false-positive results when adopting the Ames test or other methods to estimate the mutagenicity of these samples containing considerable amounts of histidine.
The mutagenicity of these traditional Chinese medicines was assayed with the new method.The RRFs of tester strains in all test groups were not significantly beyond those in the negative control groups(t test;p＞0.05);furthermore,no dose-response relationships appeared between the RRFs of tester strains and the concentrations of traditional Chinese medicine.Thus,the results in the new method indicated that all these traditional Chinese medicines were nonmutagenic.
These traditional Chinese medicines were also proved to be nonmutagenic with mammalian bone marrow chromosome aberration test.These results were in accordance with the results in the new method,which suggested that the new method was effective to monitor the mutagenicity of traditional Chinese medicine.
3.A novel mutation detection assay based on MutS and real-time fluorescent quantitative PCR assay
MutS is known to be an important component of mismatch repair(MMR) systems and play a vital role in maintaining the fidelity of genetic information in living cells. In vivo,it specifically recognizes and binds all the base pair mismatches and some small-scale insertion/deletion mutations.However,under in vitro conditions,MutS does not bind specifically to heteroduplex DNA.Whether MutS binds to homoduplex DNA or not is closely correlated with the molar ratio of MutS to DNA.
A novel,sensitive mutation detection strategy was developed by performing real-time fluorescent quantitative PCR assay and utilising the binding property of MutS.The mutants as low as 2 percent among the normal cells can be detected with this method and this method can be applied to detect the unknown mutations within the whole genome.
4.Establishing a detection method for low-proportion mutants
A variety of methods such as DNA sequencing and SSCP for detecting DNA fragment polymorphism and mutation were established,but these methods can not be adopted to detect low-proportion mutants for lacking of high sensitivity.
Some other methods such as DNA chip can be used to detect the low-proportion mutants,but these methods are not widely adopted because of high costs.In this study, based on the difference in the denaturation temperatures between heteroduplex DNA and homoduplex DNA,a novel PCR procedure was proposed to improve the relative and absolute contents of mutant.Afterwards,samples were analyzed by single-strand conformation polymorphism with capillary electrophoresis and laser-induced fluorescence detector(CE-LIF-SSCP).Some low-proportion mutants were believed to be monitored by this method.
Ames测试是通过检测化合物能否增加组氨酸缺陷型沙门氏菌的回复突变来推测其潜在致癌性,该测试对化合物的致突变性、潜在致癌性易于测试、判断;但对于含组氨酸的复杂混合物,经典的Ames测试并不适合,检测、分析方法的不当即会造成“以假作真”的错误推断。近年来,在检测中药制剂的致突变性时,这一问题又恰恰发生了;当然,这类问题同样存在于用Ames测试来检测其它复杂混合物的致突变性时。
不久前,国家发展改革委发布了《高技术产业化“十一五”规划》,规划提出在“十一五”期间我国将实施16个高技术产业化重大专项,其中包括“现代中药”重大专项。然而,能否准确地检测、评定中药的致突变性、潜在致癌性会直接影响国家对中药产业的政策,同时也关系到中药能否被充分利用,更是关系到人类健康的头等大事。因此,建立一个适于分析复杂混合物致突变性的方法就显得尤为迫切和重要。
以表型为检测指标的突变检测方法往往会受到多种因素的影响,同时环境因子对DNA的致突变作用也绝不仅仅局限于某个特定的基因(如Ames测试中组氨酸操纵子的某个基因),这涉及到随机性还是特异性的问题,这一点在理论上至今还没有确切的答案;而直接以DNA为检测指标的突变检测方法应是解决这类问题的关键所在。本论文的相关研究工作即在上述背景下展开,三年来取得了以下研究成果。
一、对影响Ames测试结果不确定性因素的分析
Ames测试是目前各个实验室普遍采用的检测环境来源的致突变物、潜在致癌物的通用初筛方法之一,但该测试在实验设计、实验过程及实验结果的分析上仍存在一些不确定性的因素和疑问。评估一个微生物群体的自发突变率和诱发突变率在遗传理论和实验技术上都是一个涉及多方面因素的复杂性难题,估计诱发突变率时涉及到对两个正态分布的混合分布参数的估计,它需要大量的样本,但Ames测试中样本数均较小,无法用条件矩等方法估计混合分布的参数;DNA发生突变的随机性和诱发因素的相关性也是尚未阐明的难题之一。
1.1总体上来说,Ames测试菌株只能检测出导致小规模突变的致突变物和理化因子,对于更多其它类型的致突变物和理化因子则无法检出,这就导致了该测试在有效检出的致突变物类型及种类上存在较大的局限性,且易出现假阴性结果。
1.2 Ames测试菌株只能检测出发生在特定位点上的突变,发生在其它位点上的突变则无法检出。
1.3 Ames测试建立在营养贫瘠的培养基上,当用其检测复杂混合物如中药的致突变性时,可能产生假阳性结果,适应性突变更加剧了这种可能性。
1.4 Ames测试中可统计的回复子菌落数是培养时间的函数,肉眼可见的回复子菌落数随培养时间的延长而增多,人为规定的48 h或其它时间是一个“约定俗成”的惯例,缺乏其应具有的科学内涵。
1.5 Ames测试中各类型的回复子在选择培养基上的生长速率各不相同。在统计时,相当数量的生长慢的回复子因未能形成肉眼可见的回复子菌落而不能被统计,这也是Ames测试这类基于试验组与对照组间回复子菌落数差异的大小来判定物质致突变性的检测方法的致命缺陷之一。
分子生物学技术的发展使直接以DNA为检测指标的物质致突变性检测方法得以实现,其可避免Ames测试中较多不确定性因素和结果。
二、建立了一个检测中药制剂潜在致癌性的方法
Ames测试建立在含微量组氨酸的固体培养基上,要求试验组与对照组中组氨酸含量相同,以使得试验组与对照组中测试菌的分裂次数、生长量均趋于相同。该测试在检测中药致突变性时,因受中药中组氨酸含量的影响,试验组中组氨酸含量高于对照组而使对照组失去了可比性,致使多种中药在该测试中呈阳性。本研究用Ames测试检测了7种中药的致突变性,所有测试的中药均呈阳性。
Ames测试菌株在LB培养基中培养时,其生长量及瞬时生长速率不受培养基中组氨酸浓度的影响;处于衰亡期的Ames测试菌株TA100和TA98的相对回复突变频率与测试体系中诱变剂的浓度间存在剂量效应关系。基于上述的结果,建立一个根据试验组与对照组中衰亡期的测试菌的相对回复突变频率的差异性来评定物质致突变性的方法。中药所含的组氨酸不会影响该测试结果的准确性,因而适于检测中药的致突变性,进而推断中药的潜在致癌性。
通过测定中药中组氨酸含量并结合统计学分析,证实这些中药的“致突变性”源于中药所含组氨酸导致的假阳性结果。应用新建方法测定了在Ames测试中呈“阳性”中药的致突变性,在该测试中试验组中测试菌的相对回复突变频率与中药浓度间不存在剂量效应关系,并且试验组与阴性对照组中测试菌的相对回复突变频率无显著性差异(t-test;p＜0.05),说明这些中药在该测试中呈阴性,即没有致突变性,进而推断这些中药没有潜在致癌性。
三种中药(银杏叶、猫眼草、三棱)进行了哺乳动物骨髓细胞染色体畸变试验,结果显示所测试中药均无致突变性,该测试结果进一步佐证了新建方法检测中药致突变性的有效性,也说明该方法适于评价中药的潜在致癌性。
三、改进了应用MutS蛋白检测突变的方法
MutS蛋白在体外能识别并结合含有错配碱基的DNA片段,多种基于此属性的突变检测方法应运而生。本文中实验结果证实,MutS蛋白在高剂量下亦能很好地结合纯合双链DNA,并且这种结合具有剂量依赖性;在低剂量下,MutS蛋白结合杂合双链DNA的能力显著强于其对纯合双链DNA的结合。
本研究中摒弃了测序法和代表性差异分析法,而采用能有效区别样品间DNA量的微小差异的实时荧光定量PCR技术来检测突变,直接比较实验样品与对照样品的Ct值的差异来判断、检测突变。实时荧光定量PCR技术的应用大大简化了检测突变的过程。
另外,证实了用此种方法能够检测在背景细胞中低至2%的突变体,是一种改进的、简便的、在DNA水平上检测突变的方法。
四、极低比例的未知突变体检测方法的探讨及初步建立
多种检测DNA片段多态性和突变的方法例如测序法及单链构象多态性(SSCP)只能检测出群体中高比例的突变体(＞9%),对于更低比例的突变体则无法有效检出。
用于检测低比例的突变体的方法需要具有高分辨力和高灵敏度,目前满足这两个条件的检测方法如芯片、生物电极等又因成本过高而难以被普遍地应用。本文设计了一个可提高群体中极低比例的突变体的相对及绝对含量的PCR程序,应用此程序提高了突变体的相对及绝对含量后,用激光诱导荧光毛细管电泳-单链构象多态性分析了实验样品及对照样品经PCR扩增后的产物。实验结果显示,在毛细管电泳谱图中,实验样品的扩增产物比对照样品明显地多出了一个峰,此峰是实验样品中极低比例的未知突变体(10~(-5))经扩增后被检测到。此方法具有一定的可行性,但还需进一步优化条件以提高突变体的检出效率,在此基础上建立能直接根据DNA的变化来检测物质致突变性的方法。
Cancer has been one of the greatest threats to human health;despite the mechanism of its occurrence and formation has not yet been fully explained,a consensus has been already obtained that most cancers are caused due to some environmental factors. Some environmental factors can change the expression levels of some cell growth factors and their receptors,which leads to the increase in mutation rates of DNA and occurrence of cancer cells.Therefore,the investigation on the ability of environmental factors to induce gene mutation is an effective way to estimate their potential to cause cancer.From the mathematical point of view,this involves the probability and statistical inference,that is,the possibility of an incident is estimated based on a seriety of experiment results and data.It is crucial for decreasing the chance of misjudgment to adopt the appropriate detection and analysis methods,and the results from inappropriate methods will inevitably lead to wrong misjudgments.
The Ames test is based on the principle that mutagenic compounds can increase the reversion frequencies of histidine-deficient strains,thereby speculating their potential carcinogenicity.The potential carcinogenicity of pure compounds can be well estimated with the Ames test;however,as to complex mixtures,it is inappropriate to adopt the Ames test to monitor their potential carcinogenicity.Similarly,it is inappropriate to adopt the Ames test to monitor the potential carcinogenicity of traditional Chinese medicine because traditional Chinese medicine is a mixture,too.
Not long ago,the National Development and Reform Commission issued the high-tech industries development during 11th five-year plan,16 major projects including the 'modem traditional Chinese medicine' project will be implemented.So, whether the mutagenicity of traditional Chinese medicine can be accurately determined will have direct affects on not only the national policy on the traditional Chinese medicine industry,but also the utilization of traditional Chinese medicine. Therefore,it is necessary to develop a novel mutagenicity detection method for the complex mixtures including traditional Chinese medicine.
The bacterial reversion assays such as the Ames test were affected by multiple factors;at the same time,the environmental factors can change not only a specific gene but also many other genes,which is related to specificity or random.Therefore, the mutation detection method at DNA level may be a way to solve the above issues.
The study in this paper started with these problems mentioned above,and the following results were obtained in the past three years.
1.Analysis of the factors leading to uncertainty of Ames test results
The Ames test is used world-wide as an initial screen to determine the mutagenic potential of new chemicals and drugs;however it involves many uncertain factors. The estimation of the spontaneous and induced mutation rate of a microbial group is complex from the point of view of the genetic theory and experimental technique, which involves a number of factors.The estimation of induced mutation rate is related to the mixing of two normal distributions,which needs lots of samples;unfortunately, the number of samples in the Ames test is small,as a result the parameter of the mixed distribution can not be estimated with conditional moment closure model.
1.1 Only small-scale mutations such as base substitution and one or two nucleotides
insertion or deletion can be detected by the tester strains used in the Ames test,as a result many other types of mutation can not be monitored.
1.2 Only the mutations occurring at specific locuses such as hiG46 locus can be monitored,many more mutations occurring at other locuses can not be detected with the Ames test.
1.3 The Ames test is based on the minimal medium,which made this method unsuitable for the detection of mutagenicity of nutrient-rich materials. Additionally,this barren environment might also lead to the adaptive mutation, which much more enhanced the uncertainty of test results.
1.4 The revertant colony numbers are the function of incubation time,so the time to count the revertant colony numbers(48 h) is relatively arbitrary,lacking of scientific connotation.
1.5 Various types of revertants grew at different rates in the selective medium; thereby the revertants with a higher growth rate were probably counted compared with those with lower growth rates.That is to say,not all the revertants could be counted,which was a vital defect to the Ames test.
2.A novel potential eareinogenieity detection method for traditional Chinese medicine
The Ames test is based on the test system that contains trace amounts of histidine (15.52μg/plate),and the amounts ofhistidine in the test groups and the control groups are same.Many traditional Chinese medicines were estimated to be mutagenic with the Ames test,but these results on the mutagenicity of these traditional Chinese medicines might be questionable due to considerable amounts of histidine in them. Several traditional Chinese medicines were estimated with the Ames test,and all of them were found to be mutagenic.However,these samples were nonmutagenic after deducting the revertant numbers resulting from the histidine in the samples.
Additional excessive external histidine did not affect the growth of TA100 when TA100 was cultured in LB broth;furthermore,dose-response relationships occurred between the mutagen concentrations and the relative reversion frequencies of the tester strains during the declining phase.According to these results,a novel strategy based on liquid LB broth to estimate the mutagenicity of samples containing much free and/or protein-bound histidine was developed.This strategy could be expected to avoid the false-positive results when adopting the Ames test or other methods to estimate the mutagenicity of these samples containing considerable amounts of histidine.
The mutagenicity of these traditional Chinese medicines was assayed with the new method.The RRFs of tester strains in all test groups were not significantly beyond those in the negative control groups(t test;p＞0.05);furthermore,no dose-response relationships appeared between the RRFs of tester strains and the concentrations of traditional Chinese medicine.Thus,the results in the new method indicated that all these traditional Chinese medicines were nonmutagenic.
These traditional Chinese medicines were also proved to be nonmutagenic with mammalian bone marrow chromosome aberration test.These results were in accordance with the results in the new method,which suggested that the new method was effective to monitor the mutagenicity of traditional Chinese medicine.
3.A novel mutation detection assay based on MutS and real-time fluorescent quantitative PCR assay
MutS is known to be an important component of mismatch repair(MMR) systems and play a vital role in maintaining the fidelity of genetic information in living cells. In vivo,it specifically recognizes and binds all the base pair mismatches and some small-scale insertion/deletion mutations.However,under in vitro conditions,MutS does not bind specifically to heteroduplex DNA.Whether MutS binds to homoduplex DNA or not is closely correlated with the molar ratio of MutS to DNA.
A novel,sensitive mutation detection strategy was developed by performing real-time fluorescent quantitative PCR assay and utilising the binding property of MutS.The mutants as low as 2 percent among the normal cells can be detected with this method and this method can be applied to detect the unknown mutations within the whole genome.
4.Establishing a detection method for low-proportion mutants
A variety of methods such as DNA sequencing and SSCP for detecting DNA fragment polymorphism and mutation were established,but these methods can not be adopted to detect low-proportion mutants for lacking of high sensitivity.
Some other methods such as DNA chip can be used to detect the low-proportion mutants,but these methods are not widely adopted because of high costs.In this study, based on the difference in the denaturation temperatures between heteroduplex DNA and homoduplex DNA,a novel PCR procedure was proposed to improve the relative and absolute contents of mutant.Afterwards,samples were analyzed by single-strand conformation polymorphism with capillary electrophoresis and laser-induced fluorescence detector(CE-LIF-SSCP).Some low-proportion mutants were believed to be monitored by this method.
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